Project Summary/Abstract Regulatory T cells (Treg) are a distinct subset of immune cells that play a critical role in properly maintaining immune homeostasis. Indeed, changes in Treg homeostasis or function is highly implicated in a variety of autoimmune diseases settings as well as in cancer, suggesting clinical importance of understanding Treg biology. Despite decades of research on Tregs, the molecular basis of how Tregs suppress unwanted immune responses are unclear. The hurdle mainly comes from ?too many? mechanisms proposed as to how Tregs function. Here, we propose a new paradigm for the function of Tregs with the central hypothesis that Tregs impose a ?core? suppressive program in target effector CD4 T cells (Teff) at the level of ?translation,? the process where proteins are generated from the genetic information encoded in the format of RNA. Although proteins are the biomolecules that confer most biological function, it is surprising that the steady-state abundance of mRNA known as the transcriptome is usually the end-point analysis chosen to explain mechanisms of immune responses. We believe this gap comes from the lack of precise tools to interrogate the translatome (all the mRNA transcripts that are translated in a given moment) in primary immune cells. We repurposed the RiboTag genetic tool that introduces an epitope-tag in a core ribosomal protein to study the translatome of primary immune cells by translating ribosome affinity purification (TRAP). Using this tool, we propose to follow up on our preliminary observation that indeed Tregs are necessary and sufficient to regulate the overall protein synthetic output in Teff cells both in vitro and in vivo. In Aim1, we will interrogate the genome-wide changes in ribosome-bound mRNA transcripts during Treg encounter. We will then characterize cis-regulatory elements (sequence within the mRNA molecule) in the specific mRNA transcripts using bioinformatics analysis to understand the mechanism of such specificity. In Aim2, we will explore the hypothesis that trans-acting protein factors associated with the ribosome (riboproteome) itself is under dynamic regulation during Treg encounter. Using the same genetic tool that allows direct ribosome immunoprecipitation, we will interrogate the entire proteome of such bound factors using mass spectrometry analysis. Delineation of the logic behind how specific mRNAs are chosen in time and space to become functional protein molecules may revolutionize the way we view gene expression control in immunity.